Polyhydroxy diamine surfactants and their use in gene transfer

ABSTRACT

The use of carbohydrate-based surfactant compounds having the general formula (I):                  
 
wherein Y 1  and Y 2 , which may be the same or different, are carbohydrate groups;
     R 1  and R 2 , which may be the same or different, are selected from:
       a) hydrogen;   b) C (1-24)  alkyl group;   c) C (1-24)  alkyl carboxy group; or   d) a carbon chain of 2 to 24 carbon atoms having one or more carbon/carbon double bonds,   
       and n is from 1 to 10;   for facilitating the transfer of DNA or RNA polynucleotides, or analogs thereof, into a eukaryotic or prokaryotic cell in vivo or in vitro.   

     New carbohydrate-based surfactant compounds are also disclosed.

NEW USE

This invention relates to new uses for carbohydrate-based surfactantcompounds. Such uses include facilitating the transfer of compounds intocells for drug delivery and facilitating the transfer ofoligonucleotides and polynucleotides into cells for gene expressionstudies or gene therapy. The invention also relates to newcarbohydrate-based surfactant compounds and methods for theirproduction.

Surfactants are substances that markedly affect the surface propertiesof a liquid, even at low concentrations. For example surfactants willsignificantly reduce surface tension when dissolved in water or aqueoussolutions and will reduce interfacial tension between two liquids or aliquid and a solid. This property of surfactant molecules has beenwidely exploited in industry, particularly in the detergent and oilindustries. In the 1970s a new class of surfactant molecule wasreported, characterised by two hydrophobic chains with polar heads whichare linked by a hydrophobic bridge (Deinega, Y et al., Kolloidn. Zh. 36,649, 1974). These molecules, which have been termed “gemini” (Menger, FM and Littau, C A, J. Am. Chem. Soc. 113, 1451, 1991), have verydesirable properties over their monomeric equivalents. For example theyare highly effective in reducing interfacial tension between oil andwater based liquids and have a very low critical micelle concentration.Recently, Pestman et al have reported the synthesis and characterisationof nonionic carbohydrate-based gemini surfactants (Pestman, J M et al,Langmuir, 13, 6857–6860, 1997).

Cationic surfactants have been used inter alia for the transfection ofpolynucleotides into cells in culture, and there are examples of suchagents available commercially to scientists involved in genetictechnologies (for example the reagent Tfx™-50 for the transfection ofeukaryotic cells available from Promega Corp. WI, USA).

The efficient delivery of DNA to cells in vivo, either for gene therapyor for antisense therapy, has been a major goal for some years. Muchattention has concentrated on the use of viruses as delivery vehicles,for example adenoviruses for epithelial cells in the respiratory tractwith a view to corrective gene therapy for cystic fibrosis (CF).However, despite some evidence of successful gene transfer in CFpatients, the adenovirus route remains problematic due to inflammatoryside-effects and limited transient expression of the transferred gene.Several alternative methods for in vivo gene delivery have beeninvestigated, including studies using cationic surfactants. Gao, X etal. (1995) Gene Ther. 2, 710–722 demonstrated the feasibility of thisapproach with a normal human gene for CF transmembrane conductanceregulator (CFTR) into the respiratory epithelium of CF mice using aminecarrying cationic lipids. This group followed up with a liposomal CFgene therapy trial which, although only partially successful,demonstrated the potential for this approach in humans (Caplen, N J. etal., Nature Medicine, 1, 39–46, 1995). More recently other groups haveinvestigated the potential of other cationic lipids for gene delivery,for example cholesterol derivatives (Oudrhiri, N et al. Proc. Natl.Acad. Sci. 94, 1651–1656, 1997). This limited study demonstrated theability of these cholesterol based compounds to facilitate the transferof genes into epithelial cells both in vitro and in vivo, therebylending support to the validity of this general approach.

These studies, and others, show that in this new field of research thereis a continuing need to develop novel low-toxicity surfactant moleculesto facilitate the effective transfer of polynucleotides into cells bothin vitro for transfection in cell-based experimentation and in vivo forgene therapy and antisense treatments. The present invention seeks toovercome the difficulties exhibited by existing compounds.

The invention relates to the use of carbohydrate-based surfactantcompounds having the general formula (1):

wherein Y₁ and Y₂, which may be the same or different, are carbohydrategroups, preferably sugars;

-   R₁ and R₂, which may be the same or different, are selected from:    -   a) hydrogen;    -   b) C₍₁₋₂₄₎ alkyl group;    -   c) C₍₁₋₂₄₎ alkyl carboxy group; or    -   d) a carbon chain of 2 to 24 carbon atoms having one or more        carbon/carbon double bonds,-   and n is from 1 to 10;-   or a salt, preferably a pharmaceutically acceptable salt thereof,-   for facilitating the transfer of DNA or RNA polynucleotides, or    analogs thereof, into a eukaryotic or prokaryotic cell in vivo or in    vitro.

Preferably the compound is symmetrical, that is the groups R₁ and R₂ arethe same, and Y₁ and Y₂ are the same. The molecular symmetry allowsthese compounds to be referred to as “gemini” surfactants.

In a preferred embodiment, the carbohydrate groups Y₁ and Y₂ are sugars,attached to the nitrogen via a reduced imine bond. Such sugars includemonosaccharides such as glucose and fructose, disaccharides such aslactose and more complex sugars, for instance sugars based on cellulose.

In a particularly preferred embodiment, Y₁ and Y₂ are glucose; thecompounds having the general structure of formula (II):

wherein Glu is glucose in open chain form (glucitol) linked via the C-1(aldehyde carbon), and R₁, R₂ and n are as hereinbefore defined.

In a further preferred embodiment R₁ and R₂ are alkyl groups ofchain-length C₍₁₀₋₂₀₎, most preferably C₍₁₂₋₁₈₎, and n is between 2 and8, most preferably 4 or 6.

In a still further preferred embodiment R₁ and R₂ are C₍₁₂₋₂₄₎,preferably C₍₁₆₋₂₀₎, most preferably C₁₈ carbon chains having one ormore carbon/carbon double bonds.

Such compounds are new and form part of the present invention.

The present invention shows the surprising finding thatcarbohydrate-based surfactants are highly efficient agents forfacilitating the transfection of polynucleotides into cells.

Compounds of formula (1) in which R₁ and R₂ are not both C₍₁₋₂₄₎ alkylcarboxyl groups are new. Accordingly, in a further aspect, the presentinvention provides for compounds of formula (I) in which one of R₁ or R₂is an alkyl group of chain-length C₍₁₋₂₄₎, and the other is a C₍₁₋₂₄₎alkyl carboxy group.

Compounds of the present invention may be prepared from readilyavailable starting materials using synthetic chemistry well known to theskilled person. A general process for preparing carbohydrate-basedsurfactant compounds comprises the addition of carbohydrate groups atthe amine ends of an alkyl diamine compound. The following is a generalscheme (scheme 1) for the synthesis of the sugar-based compounds of theinvention, as illustrated for glucose-based compounds:

For R₁=C₍₁₋₂₄₎ alkyl carboxy, the second step will be the formation ofan amide bond, using a suitable acylating agent, for instance anactivated derivative of the corresponding acid.

Preferably the scheme for the synthesis of the sugar-based compounds ofthe invention, as illustrated for glucose-based compounds, is as shownin scheme 2:

In a further aspect, the compounds of the invention which comprisecarbon chains of 2 to 24 carbon atoms and having one or morecarbon/carbon double bonds may be prepared according to scheme 3 (FIG.3) as exemplified for the C₁₈ oleyl compound. The skilled person can usethis information to devise analogous processes for preparing othercompounds comprising carbon chains of 2 to 24 carbon atoms and havingone or more carbon/carbon double bonds.

The processeses described above are for the synthesis of symmetrical,that is “gemini”, carbohydrate-based surfactants. Non-symmetricalcarbohydrate-based surfactants of the invention can be prepared byintroducing asymmetry, for example at the primary amines of the diamine,by using different protecting groups.

In a further aspect, the carbohydrate-based surfactant compounds areused to facilitate the transfer of oligonucleotides and polynucleotidesinto cells to achieve an antisense knock-out effect, for gene therapyand genetic immunisation (for the generation of antibodies) in wholeorganisms. In a further preferred embodiment, the carbohydrate-basedsurfactant compounds are used to facilitate the transfection ofpolynucleotides into cells in culture when such transfer is required,in, for example, gene expression studies and antisense controlexperiments among others. The polynucleotides can be mixed with thecompounds, added to the cells and incubated to allow polynucleotideuptake. After further incubation the cells can be assayed for thephenotypic trait afforded by the transfected DNA, or the levels of mRNAexpressed from said DNA can be determined by Northern blotting or byusing PCR-based quantitation methods for example the Taqman™ method(Perkin Elmer, Connecticut, USA). Compounds of the invention offer asignificant improvement, typically between 3 and 6 fold, in theefficiency of cellular uptake of DNA in cells in culture, compared withcompounds in the previous art. In the transfection protocol, the geminicompound may be used in combination with one or more supplements toincrease the efficiency of transfection. Such supplements may beselected from, for example:

-   (i) a neutral carrier, for example dioleyl phosphatidylethanolamine    (DOPE) (Farhood, H., et al (1985) Biochim. Biophys. Acta 1235 289);-   (ii) a complexing reagent, for example the commercially available    PLUS reagent (Life Technologies Inc. Maryland, USA) or peptides,    such as polylysine or polyornithine peptides or peptides comprising    primarily, but not exclusively, basic amino acids such as lysine,    ornithine and/or arginine. The list above is not intended to be    exhaustive and other supplements that increase the efficiency of    transfection are taken to fall within the scope of the invention.

In still another aspect, the invention relates to the transfer ofgenetic material in gene therapy using the compounds of the invention.

Yet another aspect of the invention relates to methods to effect thedelivery of non-nucleotide based drug compounds into cells in vitro andin vivo using the compounds of the invention.

In a further aspect, the invention relates to methods to facilitate thetransfer of a polynucleotide or an anti-infective compounds intoprokaryotic or eukaryotic organism for use in anti-infective therapy.

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications have been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Transfection” refers to the introduction of polynucleotides into cellsin culture using methods involving the modification of the cell membraneeither by chemical or physical means. Such methods are described in, forexample, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989). The polynucleotides may be linear or circular, single-strandedor double-stranded and may include elements controlling replication ofthe polynucleotide or expression of homologous or heterologous geneswhich may comprise part of the polynucleotide.

The invention will now be described by way of the following examples.

EXAMPLE 1 Transfection of Recombinant Plasmid Expressing Luciferase intoCells in Culture Using Carbohydrate-Based Surfactant Compounds

Carbohydrate-based surfactant compounds having the general structure offormula (II)

were synthesised according to the method as hereinbefore described. Thefollowing compounds were made:

Compound no. R₁-n-R₂ GS_G_1: 16-6-16 GS_G_2: 18-6-18 (unsaturated(oleyl) R chains) GS_G_3: 12-6-12 GS_G_4: 14-6-14 GS_G_5: 14-4-14GS_G_6: 16-4-16 GS_G_7: 12-4-12 GS_G_8: 18-4-18 GS_G_9: 18-6-18

Transfection studies were performed using the adherent cell line CHO-K1(Puck et al. 1958). Complete medium consisted of MEM alpha mediumsupplemented with 10% v/v foetal bovine serum and 1× L-Glutamine. Allmedia and supplements were obtained from Life Technologies.

Stable transfected cell lines expressing β-galactosidase were generatedby cotransfection of the plasmid pSV-β-Galactosidase Control Vector(Promega) with the plasmid Selecta Vecta-Neo (R & D Systems) in a 10:1ratio. Following G418 (Life Technologies) selection (0.8 mg ml⁻¹),candidate cell lines were tested for β-galactosidase activity (β-GalReporter Gene Assay, chemiluminescent; Roche Diagnostics).

In Vitro Gene Transfection.

Cells were seeded into 96-well plates (Beckton Dickinson) 16–18 hoursprior to transfection at an approximate density of 1×10⁴ cells per well.For transfection, 64 ng of the luciferase reporter gene plasmid,pGL3-Control Vector (Promega) per well, was incubated with variousconcentrations of the carbohydrate-based gemini compounds. After 30minutes incubation at RT, OPTI-MEM™ medium (Life Technologies) was addedto the transfection mixture and the solution placed on the cells (finalvolume per well: 100 μl). Following a 3 hour or over night incubation at37° C., the transfection solution was replaced with complete medium andthe cells incubated further at 37° C. Reporter gene assays wereperformed according to the manufacturer's guidelines (Roche Diagnostics)approximately 48 hours post transfection. Luminescence was measured in aPackard TopCount NXT Microplate Scintillation and Luminescence Counter.For normalization purpose, β-galactosidase activity (β-Gal Reporter GeneAssay, chemiluminescent; Roche Diagnostics) was measured and luciferaseactivity per β-galactosidase activity was calculated. The results areshown in FIGS. 1 and 2.

EXAMPLE 2 Transfection Efficiency of GS_G_(—)2 in the Presence orabsence of foetal Calf Serum (FCS)

GS_G_(—)2 was prepared as described hereinabove and used in experimentsto test the transfection efficiency of the compound as described inexample 1. Two experiments were conducted, in both experiments thecompound was tested at 4 uM, 8 uM, 10 uM, 20 uM and 30 uM both in thepresence and absence of PLUS reagent. In the first experiment the CHO-K1cells were incubated overnight without foetal calf serum (FCS) and inthe second experiment the CHO-K1 cells were incubated overnight in thepresence of FCS. The results showed that preincubation with foetal calfserum had no effect on the transfection efficiency of the GS_G_(—)2compound. This result was surprising as it is well known in the art thatserum reduces transfection efficiency. The presence or absence of PLUSreagent had no significant effect on transfection efficiency in eitherexperiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Transfection of CHO-K1 cells (stable transfected withbeta-galactosidase) with carbohydrate-based gemini compounds GS-G-3,GS-G4, GS-G-5, GS-G-6, GS-G-7, GS-G-8, and GS-G-9. Concentrations of thecompounds in μM is shown on the x axis. Bars represent the mean cps(counts per second) of 8 experiments±the standard error of the mean.

FIG. 2. Transfection of CHO-K1 cells (stable transfected withbeta-galactosidase) with carbohydrate-based gemini compound GS-G-1.Concentrations of the compound in μM is shown on the x axis. Barsrepresent the mean cps (counts per second) of 8 experiments±the standarderror of the mean.

FIG. 3. Scheme 3 shows a general process for the preparation of an oleylcompound of the invention.

1. A method of transferring a DNA or RNA polynucleotide into aeukaryotic cell in vivo or in vitro, the method comprising contactingthe cell with a DNA or RNA polynucleotide and a compound of formula (I):

wherein Y₁ and Y₂, which may be the same or different, are carbohydrategroups; R₁ and R₂, which may be the same or different, are selected fromthe group consisting of: hydrogen, C_((1–24)) alkyl group, C₍₁₋₂₄₎ alkylcarboxy group, and a carbon chain of 2 to 24 carbon atoms having one ormore carbon/carbon double bonds; and n is from 1 to 10; or apharmaceutically acceptable salt thereof.
 2. The method of claim 1wherein the carbohydrate groups Y₁ and Y₂ are sugars.
 3. The method ofclaim 1 wherein R₁ and R₂ are alkyl groups of chain-length C₍₁₀₋₂₀₎ andn is between 2 and
 8. 4. The method of claim 3 wherein R₁ and R₂ arealkyl groups of chain-length C₍₁₂₋₁₈₎ and n is 4 or
 6. 5. The method ofclaim 1 wherein R₁ and R₂ are carbon chains of 2 to 24 carbon atomshaving one or more carbon/carbon double bonds.
 6. The method of claim 5wherein the carbon chains have 18 carbon atoms.
 7. The method of claim 1wherein the compound is symmetrical, that is the groups R₁ and R₂ arethe same, and Y₁ and Y₂ are the same.
 8. The method of claim 1 whereinthe polynucleotide is transferred into the cell in culture.
 9. Acompound of formula (I):

wherein Y1 and Y2, which may be the same or different, are carbohydrategroups; one of R1 and R2 is selected from the group consisting ofhydrogen, a C₍₁₋₂₄₎ alkyl group, a C₍₁₋₂₄₎ alkylcarboxy group, and acarbon chain of 2 to 24 carbon atoms having one or more carbon/carbondouble bonds; the other of R1 and R2 is selected from the groupconsisting of a C₍₁₋₂₄₎ alkyl group and a carbon chain of 2 to 24 carbonatoms having one or more carbon/carbon double bonds; and n is from 1 to10; or a pharmaceutically acceptable salt thereof.
 10. The compound ofclaim 9 wherein R₁ and R₂ are alkyl groups of chain-length C₍₁₀₋₂₀₎ andn is between 2 and
 8. 11. The compound of claim 9 wherein R₁ and R₂ areeach oleyl, C₁₂-alkyl, C₁₄-alkyl, C₁₆-alkyl, or C₁₈-alkyl; Y₁ and Y₂ areeach glucitol; and n is 4 or
 6. 12. The compound of claim 9 wherein thecompound is a gemini compound where R₁ and R₂ are the same and Y₁ and Y₂are the same.
 13. The compound of claim 12 which has the formula (II):

wherein Glu is glucose in open chain form (glucitol).
 14. The compoundof claim 9 wherein one of R₁ and R₂ is an alkyl group of chain-lengthC₍₁₋₂₄₎, and the other of R₁ and R₂ is a C₍₁₋₂₄₎ alkyl carboxy group.15. The compound of claim 9 wherein R₁ and R₂ are carbon chains of 2 to24 carbon atoms having one or more carbon/carbon double bonds.
 16. Thecompound of claim 15 wherein the carbon chain has 18 carbon atoms.